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CD18 in Monogenic and Polygenic Inflammatory Processes of the Skin

      The β2 integrin family (CD11/CD18) of leukocyte adhesion molecules plays a key role in inflammation. Absence of the common β chain (CD18) leads to leukocyte adhesion deficiency-1 (LAD1) in humans. We here summarize data of two genetically defined mice models of β2 integrin deficiency, one with a CD18 null mutation (CD18−/−), and the other one with a hypomorphic CD18 mutation (CD18hypo). Firstly, we focus on the underlying mechanism of a severely impaired wound healing in CD18−/− mice, outlining a scenario in which a defective extravasation and phagocytosis of CD18−/− neutrophils results in delayed myofibroblast-dependent wound contraction owing to a deficient transforming growth factor-β1 release. Based on this, we have identified a potential therapy that fully rescued the impaired wound healing in CD18−/− mice. Secondly, we expand on a CD18hypo PL/J mouse model closely resembling human psoriasis. Apart from common clinical and pathophysiological features, this psoriasiform dermatitis also depends on the presence of activated CD4+T cells. We here recapitulate the influence of a reduced CD18 gene expression on T-cell function, also with regard to CD18 gene–dose effects, and its contribution to the pathogenesis of this disease. Taken together, these unique features make this model a valuable tool for investigations into the pathogenesis of human psoriasis – including its polygenic base – and future preclinical studies.
      EPC
      endothelial progenitor cell
      hypo
      hypomorphic
      LAD1
      leukocyte-adhesion deficiency type-1
      PASI
      psoriasis area and severity index
      SMA
      smooth muscle actin
      TGF
      transforming growth factor
      WT
      wild type

      β2 Integrins In Inflammation

      During the last years, the genetic approach for studies on leukocyte and endothelial cell adhesion molecules has been successfully implemented (
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      ). Genetically defined mice represent models of human diseases, which allow the functional analysis of genes and of the polygenic susceptibility for inflammatory disorders such as autoimmune diseases, arthritis, psoriasis, atopic dermatitis, asthma, and atherosclerosis (
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      ). Our group has a long-standing interest in further defining the role of β2 integrins in complex skin diseases. β2 integrins are leukocyte adhesion molecules exclusively expressed on hematopoietic cells being responsible for cell–cell contacts in a variety of inflammatory interactions, and in particular, they participate crucially in the extravasation process of leukocytes (Figure 1) (
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      Structure and function of the leukocyte adhesion molecules CD11/CD18.
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      The leukocyte integrins.
      ). After tethering and rolling, they play a unique role in firm adhesion and transmigration through endothelia (
      • Springer T.A.
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      The importance of the Mac-1, LFA-1 glycoprotein family in monocyte and granulocyte adherence, chemotaxis, and migration into inflammatory sites: insights from an experiment of nature.
      ). The common β chain (CD18) associates with four α subunits (αL, αM, αX, and αD) forming distinct functional heterodimers termed LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18), and CD11d/CD18 (
      • Arnaout M.A.
      Structure and function of the leukocyte adhesion molecules CD11/CD18.
      ). These receptors interact with more than 20 ligands, of which the most prominent belong to the family of intercellular adhesion molecule (
      • Dustin M.L.
      • Springer T.A.
      Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells.
      ;
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      Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1.
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      ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).
      ;
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      Cloning and characterization of a new intercellular adhesion molecule ICAM-R.
      ;
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      Characterization of the function of intercellular adhesion molecule (ICAM)-3 and comparison with ICAM-1 and ICAM-2 in immune responses.
      ).
      Figure thumbnail gr1
      Figure 1Features of β2 integrins. The αβ heterodimeric structure is common to all integrins. The α chain includes seven extracellular N-terminal homologous repeats organized into a β propeller structure. The α chain I domain is shown in pink with the embedded metal ion-dependent adhesion site motif in orange, and the β chain I-like domain with metal ion-dependent adhesion site motif is shown in a corresponding manner. The GFFKR sequence in the cytoplasmic tail of the α subunit is involved in heterodimer assembly and regulation of ligand recognition. The heterodimer is illustrated in the “closed” or inactive state that undergoes tertiary and quaternary changes in response to inside-out signals. See “Structure and distribution,” “Ligand Recognition,” and “Inside-out Signaling” for details.
      This figure was reproduced from
      • Harris E.S.
      • McIntyre T.M.
      • Prescott S.M.
      • Zimmerman G.A.
      The leukocyte integrins.
      , with permission from The American Society for Biochemistry and Molecular Biology.
      Lack of CD18 leads to leukocyte-adhesion deficiency type-1 (LAD1), a recessively inherited disorder in which deletions, truncations, substitutions, or frameshift mutations impair β2-integrin function (
      • Kishimoto T.K.
      • Hollander N.
      • Roberts T.M.
      • Anderson D.C.
      • Springer T.A.
      Heterogeneous mutations in the beta subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency.
      ;
      • Arnaout M.A.
      Structure and function of the leukocyte adhesion molecules CD11/CD18.
      ). The severity of this disease correlates with the degree of loss of CD18 (
      • Anderson D.C.
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      • Rothlein R.
      • Miller L.J.
      • et al.
      The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features.
      ). In the absence of functional CD18, severe defects in cell–cell cooperation occur leading to a lack of homotypic lymphocyte adhesion (
      • Mentzer S.J.
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      • Burakoff S.J.
      • Martz E.
      LFA-1 membrane molecule in the regulation of homotypic adhesions of human B lymphocytes.
      ;
      • Rothlein R.
      • Springer T.A.
      The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester.
      ;
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      • de Graaff M.
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      • Meijer C.J.
      • Pals S.T.
      Induction of homotypic T cell adhesion by triggering of leukocyte function-associated antigen-1 alpha (CD11a): differential effects on resting and activated T cells.
      ) and hampered T-cell activation (
      • Davignon D.
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      Monoclonal antibody to a novel lymphocyte function-associated antigen (LFA-1): mechanism of blockade of T lymphocyte-mediated killing and effects on other T and B lymphocyte functions.
      ;
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      Distinct roles for LFA-1 and CD28 during activation of naive T cells: adhesion versus costimulation.
      ;
      • Scharffetter-Kochanek K.
      • Lu H.
      • Norman K.
      • et al.
      Spontaneous skin ulceration and defective T cell function in CD18 null mice.
      ;
      • Schönlau F.
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      • Grabbe S.
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      • Sorg C.
      • Sunderkötter C.
      In experimental leishmaniasis deficiency of CD18 results in parasite dissemination associated with altered macrophage functions and incomplete Th1 cell response.
      ) accompanied by a reduced IL-2 release (
      • Shier P.
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      • Ngo K.
      • et al.
      Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the beta2 integrin leukocyte function-associated antigen-1.
      ;
      • Zuckerman L.A.
      • Pullen L.
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      Functional consequences of costimulation by ICAM-1 on IL-2 gene expression and T cell activation.
      ;
      • Kim J.J.
      • Tsai A.
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      • et al.
      Intracellular adhesion molecule-1 modulates beta-chemokines and directly costimulates T cells in vivo.
      ;
      • Schönlau F.
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      • Grabbe S.
      • Pietz B.
      • Sorg C.
      • Sunderkötter C.
      In experimental leishmaniasis deficiency of CD18 results in parasite dissemination associated with altered macrophage functions and incomplete Th1 cell response.
      ). Patients suffering from LAD1 are typically subject to recurrent bacterial or fungal infection, peripheral leukocytosis, impaired wound healing, weak or absent vaccine response (
      • Fischer A.
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      Role of the LFA-1 molecule in cellular interactions required for antibody production in humans.
      ;
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      Regulation of antibody responses: the role of complement and adhesion molecules.
      ;
      • Anderson D.C.
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      ), and, in case of moderate β2 integrin-expression deficiency, also psoriasiform dermatitis (
      • van de Kerkhof P.C.
      • Weemaes C.M.
      Skin manifestations in congenital deficiency of leucocyte-adherence glycoproteins (CDLG).
      ).
      In the past years, we have established and/or studied two genetically defined mice models of β2 integrin deficiency, following two different mutation strategies for the CD18 locus: (1) Introduction of the neomycin resistance cassette into the splice acceptor site of exon 3 resulted in an out-of-frame mutation yielding no functional CD18 protein at all (
      • Scharffetter-Kochanek K.
      • Lu H.
      • Norman K.
      • et al.
      Spontaneous skin ulceration and defective T cell function in CD18 null mice.
      ). (2) Duplication of exons 2 and 3 by introduction of an insertion mutation in the murine CD18 gene yielded a hypomorphic mouse model of CD18 with a severe reduction in CD18 expression with only 2–16% of wild-type (WT) levels of CD18 (
      • Wilson R.W.
      • Ballantyne C.M.
      • Smith C.W.
      • Montgomery C.
      • Bradley A.
      • O'Brien W.E.
      • et al.
      Gene targeting yields a CD18-mutant mouse for study of inflammation.
      ;
      • Bullard D.C.
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      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ).

      Wound-healing Defect Of Cd18−/− Mice Owing To A Decrease In Tgf-β1 And Myofibroblast Differentiation

      Herein, we will first focus on the underlying mechanism of severely impaired wound healing in LAD1 (
      • Peters T.
      • Sindrilaru A.
      • Hinz B.
      • et al.
      Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.
      ). For this purpose, we have used the previously generated full knockout mouse model (CD18−/−), which closely reflects the clinical phenotype of patients suffering from severe-type LAD1 with spontaneously occurring erosions and wounds in mechanically stressed facial areas (
      • Scharffetter-Kochanek K.
      • Lu H.
      • Norman K.
      • et al.
      Spontaneous skin ulceration and defective T cell function in CD18 null mice.
      ). Firstly, it is of importance to define precisely the parameter and wound model (primary versus secondary wound healing, re-epithelialization versus wound contraction) employed for studies on tissue and wound repair (
      • Singer A.J.
      • Clark R.A.
      Cutaneous wound healing.
      ;
      • Werner S.
      • Grose R.
      Regulation of wound healing by growth factors and cytokines.
      ). Therefore, to analyze secondary wound healing under standardized conditions, full-thickness 5-mm punch biopsies were produced on the back of CD18−/− and WT control mice. Monitoring of wound sizes revealed a significant delay in wound healing from days 5 to 14 in CD18−/− mice compared to WT mice (Figure 2). As differences in wound sizes between WT control mice and CD18−/− mice were most pronounced from days 5 to 10 postwounding, which represents the phase of myofibroblast-mediated wound contraction, expression of myofibroblast markers was studied in granulation tissue at different stages of tissue repair by immunohistochemistry and Western blot analysis. Myofibroblasts are characterized by the expression of α-smooth muscle actin (α-SMA) (
      • Tomasek J.J.
      • Gabbiani G.
      • Hinz B.
      • Chaponnier C.
      • Brown R.A.
      Myofibroblasts and mechano-regulation of connective tissue remodelling.
      ), the actin isoform characteristic of vascular smooth muscle cells (
      • Skalli O.
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      • Trzeciak A.
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      • Gillessen D.
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      A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.
      ;
      • Serini G.
      • Gabbiani G.
      Mechanisms of myofibroblast activity and phenotypic modulation.
      ), which confers a high contractile activity to these cells, in vivo (
      • Serini G.
      • Gabbiani G.
      Mechanisms of myofibroblast activity and phenotypic modulation.
      ) and in vitro (
      • Arora P.D.
      • McCulloch C.A.
      Dependence of collagen remodelling on alpha-smooth muscle actin expression by fibroblasts.
      ;
      • Hinz B.
      • Celetta G.
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      • Gabbiani G.
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      Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.
      ,
      • Hinz B.
      • Dugina V.
      • Ballestrem C.
      • Wehrle-Haller B.
      • Chaponnier C.
      Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts.
      ). In CD18−/− mice, α-SMA expression was substantially reduced at days 5 and 7, compared to the WT controls. Besides, ED-A splice variant of fibronectin, another myofibroblast differentiation marker (
      • Serini G.
      • Bochaton-Piallat M.L.
      • Ropraz P.
      • Geinoz A.
      • Borsi L.
      • Zardi L.
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      The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1.
      ), was significantly decreased in CD18−/− mice as compared to the WT. Vimentin served as a housekeeping protein to equilibrate for equal numbers of fibroblastic cells, and did not differ in CD18−/− mice versus WT mice at these time points. Consequently, the detected reduction in myofibroblast differentiation markers reflected a decrease in the number of myofibroblastic cells in wounds of CD18−/− mice.
      Figure thumbnail gr2
      Figure 2Wound closure of full-thickness wounds is delayed in CD18−/− mice. Full-thickness (including the panniculus carnosus) excisional wounds were punched at two sites in the middle of the dorsum using 5-mm biopsy round knives. Each wound region was digitally photographed at indicated time points, and wounds areas were calculated using Adobe Photoshop software. (a) Macroscopic observation of wounds in CD18−/− and WT mice. Representative results of six wounds in each cohort are shown. (b) Wound sizes at any given time point after wounding were expressed as the percentage of initial (day 0) wound area for CD18−/− and WT mice. Results are expressed as the mean±SD (n=6). *P<0.05.
      This figure was reproduced from
      • Peters T.
      • Sindrilaru A.
      • Hinz B.
      • et al.
      Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.
      , with permission from the Nature Publishing Group.
      Besides, transforming growth factor (TGF)-β signalling seemed to be involved as TGFβRII protein levels were also decreased in CD18−/− granulation tissue at day 5 after wounding. Interestingly, TGF-β1 is one major cytokine inducing α-SMA expression, myofibroblast differentiation (
      • Desmoulière A.
      • Geinoz A.
      • Gabbiani F.
      • Gabbiani G.
      Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.
      ), and subsequent wound contraction (
      • Montesano R.
      • Orci L.
      Transforming growth factor beta stimulates collagen–matrix contraction by fibroblasts: implications for wound healing.
      ). This action of TGF-β1 depends on the presence of ED-A fibronectin in the extracellular matrix (
      • Serini G.
      • Bochaton-Piallat M.L.
      • Ropraz P.
      • Geinoz A.
      • Borsi L.
      • Zardi L.
      • et al.
      The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1.
      ). To assess TGF-β1 cytokine levels in wound tissue of CD18−/− mice versus WT mice, we performed immunohistochemistry for TGF-β1 on sections of 5- and 7-day-old wounds as well as ELISA measurements for quantification of active TGF-β1 from wound tissue lysates. As a result, there was distinctly less TGF-β1 detectable in the newly formed granulation tissue of CD18−/− mice at both of these time points after wounding, as compared to the WT controls. These data suggested that reduced amounts of TGF-β1 had accounted for the decreased numbers of α-SMA-positive myofibroblasts and the impaired wound contraction in CD18−/− mice.
      In order to prove a causal role for the TGF-β1 deficiency in the reduced wound closure of CD18−/− mice, we substituted the lacking endogenous TGF-β1 by injecting recombinant human TGF-β1 subcutaneously at four sites around the wound (
      • Ashcroft G.S.
      • Mills S.J.
      • Lei K.
      • et al.
      Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor.
      ). Mock injections were made using NaCl 0.9%. Gross evaluation and digital measurements of wound sizes revealed that the previously observed wound healing defect was fully restored in CD18−/− mice treated with recombinant TGF-β1, a finding further confirmed by the detection of α-SMA-positive myofibroblasts at wild-type levels in these mice (Figure 3). These data strongly suggested that TGF-β1 deficiency was the major cause of an impaired tissue repair in CD18−/− mice. However, the mechanism underlying this TGF-β1 deficiency needed clarification.
      Figure thumbnail gr3
      Figure 3Injection of TGF-β1 in wound margins rescues wound closure and myofibroblast differentiation in CD18−/− mice. Recombinant human TGF-β1 (“TGF”) was injected subcutaneously at four sites around the wound, allowing to infiltrate the wound margins, at a total dose of 0.45 μg per wound. Mock injections were made using only the solvent NaCl 0.9%. First, injections were carried out on day 1 after wounding, followed by further injections every second day until wounds were harvested. (a) Macroscopic observation of wounds in CD18−/−, WT mice with or without injection of TGF-β1. (b) Wound sizes were assessed at the indicated time points after wounding as described previously. Bars depict the median of each cohort. **P<0.005. (c) Paraffin-embedded granulation tissue of CD18−/− and WT mice was stained immunohistochemically for α-SMA 5 days after wounding. The bar indicates 100 μm; de, adjacent dermis; gt, granulation tissue; arrows indicate the newly formed epidermal leading edges.
      This figure was reproduced from
      • Peters T.
      • Sindrilaru A.
      • Hinz B.
      • et al.
      Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.
      , with permission from the Nature Publishing Group.
      To tackle the question which cells may have contributed to the observed TGF-β1 deficiency in wounds of CD18−/− mice, one has to consider that normal tissue repair follows a sequence of events involving clotting, inflammation, remodelling, and re-epithelialization (reviewed by:
      • Martin P.
      Wound healing – aiming for perfect skin regeneration.
      ;
      • Singer A.J.
      • Clark R.A.
      Cutaneous wound healing.
      ). Shortly after injury, first neutrophils and later macrophages invade the wound site. Although such inflammatory cells are an important source of growth factors and cytokines (
      • Werner S.
      • Grose R.
      Regulation of wound healing by growth factors and cytokines.
      ), adhesion molecules, and in particular integrins, are required for their extravasation. We and others have previously shown that in mouse models for toxic dermatitis and thioglycolate-induced peritonitis, extravasation of neutrophils depended highly on β2 integrins (
      • Mizgerd J.P.
      • Kubo H.
      • Kutkoski G.J.
      • Bhagwan S.D.
      • Scharffetter-Kochanek K.
      • Beaudet A.L.
      • et al.
      Neutrophil emigration in the skin, lungs, and peritoneum: different requirements for CD11/CD18 revealed by CD18-deficient mice.
      ;
      • Scharffetter-Kochanek K.
      • Lu H.
      • Norman K.
      • et al.
      Spontaneous skin ulceration and defective T cell function in CD18 null mice.
      ;
      • Walzog B.
      • Scharffetter-Kochanek K.
      • Gaehtgens P.
      Impairment of neutrophil emigration in CD18-null mice.
      ;
      • Grabbe S.
      • Varga G.
      • Beissert S.
      • et al.
      Beta2 integrins are required for skin homing of primed T cells but not for priming naive T cells.
      ). To investigate whether egress of leukocytes from the blood vessels may, in analogy to dermatitis, be dependent on β2 integrins also during wound healing, we set out to detect inflammatory cells infiltrating the wound area during the healing process. In a time-course analysis, wounds were harvested and stained with GR1 monoclonal antibodies for neutrophils or with F4/80 for macrophages. Subsequently, neutrophils and macrophages, which had egressed from the vessels into the interstitial tissue of the wound bed, were counted. Almost no neutrophils were detected in the granulation tissue of CD18−/− wounds, compared to high numbers of egressed neutrophils in WT wounds at 24 and 48 hours. However, similar numbers of egressed macrophages were observed at the wound sites of WT and CD18−/− mice. These data demonstrated that wounds of CD18−/− mice remained almost devoid of neutrophils, whereas infiltration by macrophages was normal (
      • Peters T.
      • Sindrilaru A.
      • Hinz B.
      • et al.
      Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.
      ).
      In secondary wound healing, TGF-β1, crucially required for normal wound closure, is released by macrophages during the engulfment of apoptotic polymorphonuclear neutrophils at the wound site (
      • Fadok V.A.
      • Bratton D.L.
      • Konowal A.
      • Freed P.W.
      • Westcott J.Y.
      • Henson P.M.
      Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.
      ;
      • McDonald P.P.
      • Fadok V.A.
      • Bratton D.
      • Henson P.M.
      Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells.
      ;
      • Taylor P.R.
      • Carugati A.
      • Fadok V.A.
      • et al.
      A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo.
      ). As we demonstrated, defective migration leads to a severe reduction of neutrophils in wounds of CD18−/− mice. Hence, we hypothesized that infiltrating macrophages might not sufficiently phagocytose apoptotic neutrophils in wounds of CD18−/− mice. Macrophages would thus be lacking their main stimulus to secrete TGF-β1. Consequently, the effect of phagocytosis of viable and apoptotic neutrophils by CD18−/− and WT macrophages was studied. We found that only apoptotic but not viable neutrophils, after being engulfed by macrophages, caused a dramatic induction in the release of TGF-β1, in line with earlier reports (
      • Fadok V.A.
      • Bratton D.L.
      • Konowal A.
      • Freed P.W.
      • Westcott J.Y.
      • Henson P.M.
      Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.
      ;
      • McDonald P.P.
      • Fadok V.A.
      • Bratton D.
      • Henson P.M.
      Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-beta in macrophages that have ingested apoptotic cells.
      ). In this context, it was an astounding finding that CD18−/− neutrophils had a higher resistance toward spontaneous apoptosis, as absent/reduced DNA laddering and annexin-V staining showed in CD18−/− compared to WT neutrophils (
      • Weinmann P.
      • Scharffetter-Kochanek K.
      • Forlow S.B.
      • Peters T.
      • Walzog B.
      A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice.
      ). To study whether, apart from the reduced extravasation of neutrophils into wound beds, release of TGF-β1 occurring during phagocytosis of neutrophils by macrophages may also be dependent on CD18, we in vitro co-cultured apoptotic CD18−/− and WT neutrophils with CD18−/− or WT macrophages. Supernatants were subjected to ELISA for TGF-β1. As a result, a time-dependent, up to 20-fold increase in active TGF-β1 release with a maximum at 24 hours was observed in co-cultures of WT apoptotic neutrophils and macrophages (
      • Peters T.
      • Sindrilaru A.
      • Hinz B.
      • et al.
      Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.
      ). Interestingly, TGF-β1 release did not exceed control levels after 24 hours in co-cultures of CD18−/− neutrophils with either WT or CD18−/− macrophages. In summary, these data indicated that a maximal release of TGF-β1 required CD18 expression on both neutrophils and macrophages, but CD18 expression on apoptotic neutrophils in particular seems to be of great importance for sustained TGF-β1 release by macrophages. With regard to the underlying pathophysiological mechanisms, the observed deficiency in TGF-β1 release in the absence of CD18 was caused indeed by a reduced phagocytotic efficacy of macrophages to engulf neutrophils owing to an impaired adhesion between phagocytotic effector and target cells (also see
      • van Spriel A.B.
      • Leusen J.H.
      • van Egmond M.
      • Dijkman H.B.
      • Assmann K.J.
      • Mayadas T.N.
      • et al.
      Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.
      ), thus attributable mainly to an impaired “physical attachment” in case adhesion molecules were absent (
      • Peters T.
      • Sindrilaru A.
      • Hinz B.
      • et al.
      Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.
      ). Potential contributions of the β2 integrin family to downstream signalling (
      • Piccardoni P.
      • Manarini S.
      • Federico L.
      • et al.
      SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells.
      ) during phagocytosis are currently being investigated.
      In summary, an impaired recruitment of neutrophils into the wound bed, an enhanced apoptotic resistance of neutrophils, and a reduced release of TGF-β1 from macrophages upon defective engulfment of apoptotic neutrophils collectively contribute to the observed deficiency of TGF-β1 in CD18−/− mice. Hence, CD18 deficiency has multiple effects on TGF-β1 availability.
      As TGF-β1 enhances angiogenesis, we also studied vessel formation in wound granulation tissues. A decrease in platelet/endothelial cell adhesion molecule-1 (CD31) expressing blood vessels in CD18−/− as compared to WT mice was observed by immunofluorescence histology of wounds in a time-course analysis (
      • Schruefer R.
      • Sulyok S.
      • Schymeinsky J.
      • Peters T.
      • Scharffetter-Kochanek K.
      • Walzog B.
      The proangiogenic capacity of polymorphonuclear neutrophils delineated by microarray technique and by measurement of neovascularization in wounded skin of CD18-deficient mice.
      ). This difference in angiogenesis could only be partly rescued by TGF-β1 injection, providing evidence that in addition to the TGF-β1 deficiency, other mechanisms may contribute to the impaired vessel formation. Because β2 integrins are strongly expressed on endothelial progenitor cells (EPCs), the role of β2 integrins for in vivo homing of EPCs in neovascularization was studied (
      • Chavakis E.
      • Aicher A.
      • Heeschen C.
      • et al.
      Role of beta2-integrins for homing and neovascularization capacity of endothelial progenitor cells.
      ). Firstly, our results showed that β2 integrins mediated the adhesive interactions of EPCs to mature endothelial cells and to extracellular matrix proteins, and were critical for chemokine-induced transendothelial migration of EPCs in vitro. Secondly, using a murine in vivo hind limb ischemia model, proximal femoral arteries were ligated and, by consequence, distal tissue nutrition and regeneration depended on new vessel formation. By transfusion of murine Sca-1+Lin hematopoietic progenitor cells from CD18−/− mice, it could be demonstrated that β2 integrins were involved in the homing of hematopoietic progenitor cells to sites of ischemia and were critical for their neovascularization capacity. This was shown quantitatively by assessing the reconstitution of vessel density and functionally by determining distal blood flow at given time points after transfusion of EPCs into recipients using color-encoded Doppler laser analysis. Interestingly, pre-activation of β2 integrins on the transfused EPC further augmented the in vivo neovascularization capacity of EPC, indicating a novel therapeutic strategy to promote homing of EPC (
      • Chavakis E.
      • Aicher A.
      • Heeschen C.
      • et al.
      Role of beta2-integrins for homing and neovascularization capacity of endothelial progenitor cells.
      ).
      In summary, CD18 is central for the emigration of neutrophils into the wound bed. Murine CD18−/− macrophages release significantly less TGF-β1 after phagocytosis of apoptotic neutrophils compared to WT macrophages. This reduced release of TGF-β1 is owing to an impaired adhesion and subsequently impaired phagocytosis of CD18−/− neutrophils by macrophages. Exclusively apoptotic neutrophils can induce TGF-β1 release from macrophages, whereas apoptosis is significantly reduced in CD18−/− neutrophils compared to WT neutrophils, owing to an enhanced bcl-x/bax-α ratio. All of these mechanisms likely contribute to the observed TGF-β1 deficiency and defective healing of acute wounds, which can be widely rescued by the local injection of recombinant TGF-β1 in CD18−/− mice. In addition, β2 integrins play a non-redundant role for homing and neovascularization capacity of EPCs.
      The herein summarized data thus demonstrates that the paracrine secretion of growth factors induced by wound-infiltrating neutrophils is essential for myofibroblast differentiation in tissue repair of cutaneous full-thickness wounds, implying a therapeutic value for growth factor substitution during specific wound healing deficiency. Furthermore, the pre-activation of β2 integrins appears to be a feasible tool to improve the efficacy of EPC-induced neovascularization, possibly relevant for patients with ischemic disease and distinct tissue repair disturbances. Regarding the importance of β2 integrins in the mobilization and migration of hematopoietic cells during tissue repair, this endorses an analogous conception as outlined also for other adhesion molecules in (a) double P- and E-selectin-deficient mice (
      • Subramaniam M.
      • Saffaripour S.
      • Van De Water L.
      • Frenette P.S.
      • Mayadas T.N.
      • Hynes R.O.
      • et al.
      Role of endothelial selectins in wound repair.
      ), (b) functionally P- and E-selectin-deficient mice (mice deficient in β-1,4-galactosyltransferase, which glycolsylates the P- and E-selectin ligands (
      • Mori R.
      • Kondo T.
      • Nishie T.
      • Ohshima T.
      • Asano M.
      Impairment of skin wound healing in beta-1,4-galactosyltransferase-deficient mice with reduced leukocyte recruitment.
      )), and (c) LAD2 patients suffering from a glycosylation deficiency owing to a gene defect in the GDP-fucose transporter (
      • Luhn K.
      • Wild M.K.
      • Eckhardt M.
      • Gerardy-Schahn R.
      • Vestweber D.
      The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter.
      ). These all have in common that affected individuals show an altered emigration of neutrophils and/or macrophages, combined with an impaired wound healing. Interestingly, intercellular adhesion molecule-1−/− mice lacking one of the multiple counter receptors of the β2 integrins only reveal a subtle wound-healing defect (
      • Nagaoka T.
      • Kaburagi Y.
      • Hamaguchi Y.
      • et al.
      Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression.
      ). However, none of these studies have directly analyzed wound contraction.

      The Role Of Cd18 In A Murine T-cell-dependent Psoriasis Model

      Another genetically defined mouse model with a specific gene defect in the CD18 locus, currently the only available polygenic mouse model for a psoriasiform skin disease, helped us to explore the involvement of distinct T-cell populations in this inflammatory disease. In addition, it will allow for identification of modifier genes and their interaction in the precipitation of this disease.
      Owing to a hypomorphic CD18 mutation, a skin disease developed in PL/J mice, which strongly resembled human psoriasis clinically, histologically, and in response to therapy (
      • Wilson R.W.
      • Ballantyne C.M.
      • Smith C.W.
      • Montgomery C.
      • Bradley A.
      • O'Brien W.E.
      • et al.
      Gene targeting yields a CD18-mutant mouse for study of inflammation.
      ;
      • Bullard D.C.
      • Scharffetter-Kochanek K.
      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ). Affected mice presented with erythema, crusts, scaling, and alopecia starting at the face and involving the trunk (Figure 4). Compared to the two-layered epidermis in WT mice, epidermal thickening occurred in CD18hypo mice, with subcorneal micro-abscesses and hyperkeratosis as well as a severe T-cell-dominated infiltrate in the dermis, all of which characteristic hallmarks of human psoriasis. Only mice homozygous for the CD18-hypomorphic (CD18hypo) mutation but not heterozygous littermate controls developed this phenotype, suggesting that the disease was recessively inherited. Interestingly, the psoriasiform skin inflammation only occurred on PL/J mice carrying the CD18hypo mutation but not on the 129Sv or C57BL/6J genetic background with the same mutation (
      • Bullard D.C.
      • Scharffetter-Kochanek K.
      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ). Hence, apart from the CD18 mutation, a small number of genes were necessary to precipitate the phenotype. No infectious agent was detected, whereas corticosteroids suppressed the psoriasiform phenotype, suggesting the involvement of an autoimmune or otherwise (sterile) inflammatory process.
      Figure thumbnail gr4
      Figure 4In vivo depletion of CD4+ T cells in CD18hypo mice. To monitor the clinical effect of CD4+ T cell depletion, neutralizing antibodies were injected intraperitaneously at a dose of 100–150 μg twice weekly. (a) A CD18hypo mouse with a severe psoriasiform dermatitis. (b) The same mouse 6 weeks after treatment with CD4+ T-cell-depleting mAbs. Depletion efficiency was evaluated by FACS analysis of peripheral blood cells from CD18hypo mice treated with the (c) isotype control mAbs or with (d) CD4+ T cell-depleting mAbs. Mouse anti-rat (MAR) IgG2b FITC mAbs were applied for the detection of residual rat anti-mCD4 mAbs, which had previously been used for depletion of CD4+ T cells. The red circle highlights the CD4+ T-cell population. Skin sections from CD18hypo mice treated with (e) isotype control mAbs or with (f) CD4+ T-cell-depleting mAbs were immunostained with mCD4 mAbs (original magnification × 400). Arrows indicate the murine full-thickness epidermis from cornified to basal layer.
      This figure was reproduced from
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      , with permission from The American Association of Immunologists.
      The pathogenic role of β2 integrins in human psoriasis is poorly understood. CD11b expression has been reported to be reduced on peripheral blood leukocytes derived from psoriasis patients (
      • van Pelt J.P.
      • Kuijpers S.H.
      • van de Kerkhof P.C.
      • de Jong E.M.
      The CD11b/CD18-integrin in the pathogenesis of psoriasis.
      ). Further experimental evidence comes from LAD1 patients with a moderately reduced CD18 expression suffering from psoriasiform skin disease (
      • van de Kerkhof P.C.
      • Weemaes C.M.
      Skin manifestations in congenital deficiency of leucocyte-adherence glycoproteins (CDLG).
      ). Linkage analyses of psoriasis families identified a region on chromosome 17 (
      • Tomfohrde J.
      • Silverman A.
      • Barnes R.
      • et al.
      Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q.
      ), including among other gene loci, intercellular adhesion molecule-2, an important ligand of CD11/CD18 heterodimers. Vice versa, polymorphisms in the CD18 gene were found predisposing to autoimmune disease either by leading to a higher ligand affinity or by increasing expression of the CD18 protein (
      • Gencik M.
      • Meller S.
      • Borgmann S.
      • Sitter T.
      • Menezes Saecker A.M.
      • Fricke H.
      • et al.
      The association of CD18 alleles with anti-myeloperoxidase subtypes of ANCA-associated systemic vasculitides.
      ;
      • Meller S.
      • Jagiello P.
      • Borgmann S.
      • Fricke H.
      • Epplen J.T.
      • Gencik M.
      Novel SNPs in the CD18 gene validate the association with MPO-ANCA+ vasculitis.
      ). Different cell types have been suspected to be the primary inducer in the pathogenesis of psoriasis (
      • van den Oord J.J.
      • de Wolf-Peeters C.
      Epithelium-lining macrophages in psoriasis.
      ;
      • Nickoloff B.J.
      The immunologic and genetic basis of psoriasis.
      ;
      • Nickoloff B.J.
      • Nestle F.O.
      Recent insights into the immunopathogenesis of psoriasis provide new therapeutic opportunities.
      ;
      • Ozawa M.
      • Aiba S.
      Immunopathogenesis of psoriasis.
      ). Increasing evidence led to the current view that T cells were the main suspects responsible for its initiation (
      • Nickoloff B.J.
      • Schroder J.M.
      • von den Driesch P.
      • et al.
      Is psoriasis a T-cell disease?.
      ;
      • Krueger J.G.
      The immunologic basis for the treatment of psoriasis with new biologic agents.
      ). Therefore, we first studied different T-cell subpopulations in the skin of CD18hypo psoriasis mice (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). Compared to WT mice, a significant increase in CD4+ T cells was found in the epidermis and the dermis of CD18hypo mice compared to WT mice. Most interestingly, effective depletion of CD4+ T cells, as proven by FACS analysis, revealed a complete resolution of the psoriasiform skin disease within 6 weeks of treatment (Figure 4). The effect of the CD4+ T-cell-depleting monoclonal antibodies was evaluated by the severity of clinical symptoms using an adapted murine psoriasis area and severity index (PASI) score (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ), as commonly used for the assessment of severity in human psoriasis. A complete resolution of the psoriasiform skin disease was observed in two mice (PASI 10 down to 1) and an almost complete resolution in one mouse (PASI 8 down to 2) treated with the anti-CD4 monoclonal antibodies. This clearance of the skin inflammation persisted for an observation period of 6 months after the last administration of depleting antibody, whereas for three further CD18hypo mice mock-treated with IgG isotype antibodies, no significant changes in the PASI scores were evident. As CD4+ T cells can exert their effects via activation of cytotoxic effector functions of CD8+ T cells, CD18hypo mice were also treated with CD8+ T-cell-depleting mAb in an analogous experimental setting (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). As monitored by the adapted PASI score, treatment with neither CD8+ T-cell-depleting monoclonal antibodies nor isotype-matched control antibodies resulted in any improvement of the psoriasiform skin disease. These results suggested that CD8+ T cells were not crucial for the pathogenesis of the inflammatory skin disease of this psoriasis-like mouse model. However, apart from CD4+ T cells, a distinct role in the pathogenesis of this skin disease was also attributed to CD8+ T cells by other investigators (
      • Barlow S.C.
      • Xu H.
      • Weaver C.T.
      • Lindsey J.R.
      • Schoeb T.R.
      • Bullard D.C.
      Development of dermatitis in CD18-deficient PL/J mice is not dependent on bacterial flora, and requires both CD4+ and CD8+ T lymphocytes.
      ), possibly on the basis of a slightly different segregation of genes during the maintenance/backcrossing of the respective mouse strain (
      • Barlow S.C.
      • Collins R.G.
      • Ball N.J.
      • Weaver C.T.
      • Schoeb T.R.
      • Bullard D.C.
      Psoriasiform dermatitis susceptibility in Itgb2(tm1Bay) PL/J mice requires low-level CD18 expression and at least two additional loci for progression to severe disease.
      ).
      To determine the activation state of T cells in affected mice, CD90+ T cells were isolated from draining lymph nodes of diseased CD18hypo and healthy WT mice (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). A significant increase in the expression of CD25 (IL-2Rα chain) as a measure of T cellular activation was detected on CD4+ but not on CD8+ T cells obtained from diseased CD18hypo mice. Because the mean expression of CD25 on CD4+ T cells was twice as high in CD18hypo as in WT mice, this suggested an increased state of activation in CD4+ T cells from CD18hypo mice. To further determine whether T cells obtained from draining lymph nodes had been primed to secrete either Th1- or Th2-type cytokines, we measured the release of IFN-γ or IL-4 after culturing with different concentrations of immobilized anti-CD3 and anti-CD28 monoclonal antibodies. CD90+ T cells from affected CD18hypo mice released up to 40-fold higher amounts of IFN-γ compared to WT mice, whereas no IL-4 could be detected in the supernatants of activated T cells of either genotype. These data pointing at a prevalence of Th1-type cytokines in CD18hypo mice was supported by studies using an extended Th1/Th2 panel for cytometric bead array detection of cytokines (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). Apart from IFN-γ, very high levels of the Th1 key cytokine IL-2 were also detected in the supernatants of CD18hypo T cells. However, the Th2 cytokine IL-10 was slightly increased. Still, these data clearly demonstrated the prevalence of Th1 cytokines in T cells isolated from the skin lesion draining lymph nodes of CD18hypo mice.
      To address the question whether total absence of CD18 may equally lead to the development of a psoriasiform phenotype in mice of the PL/J strain, a PL/J mouse line with a complete deficiency in CD18 (CD18−/−) was generated (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). Remarkably, during an observation period of more than 2 years, PL/J CD18−/− mutants (n=200) did not develop any psoriasiform skin disease. Consequently, we hypothesized that the T cells required for the psoriasiform dermatitis of CD18hypo mice may not be able to enter the skin in complete absence of CD18 adhesion molecules, thus being hampered to exert their inflammatory effector functions. As antigen-specific T cells are most likely to play a central role in the pathogenesis of psoriasis, but the responsible antigen could so far not be identified, it is difficult to study the emigration kinetics of antigen-specific T cells. Therefore, we used a model of T-cell-mediated type-IV hypersensitivity, allowing the induction of an allergic contact dermatitis under standardized conditions with oxazolone as a defined antigen (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). To differentiate clearly between the deliberately induced allergic contact dermatitis and the spontaneously developing psoriasiform skin inflammation in CD18hypo PL/J mice, we only analyzed clinically healthy CD18hypo mice, comparing these mice with equally treated CD18−/− and WT mice as controls. As a result, allergic contact dermatitis could only be induced in CD18hypo and WT mice but not in CD18−/− mice upon repeated oxazolone challenge. As hematoxylin and eosin and immunostainings of sections from oxazolone-challenged ears revealed, this was owing to a failure of CD18−/− T cells, both CD4+ and CD8+, to emigrate from the vessels into the tissue, thus causally contributing to the unresponsiveness of CD18−/− mutants to oxazolone. Interestingly, T cells isolated from the draining lymph nodes of sensitized CD18−/− mice could rescue the ear swelling phenotype in response to oxazolone challenge when directly injected in the ears of CD18−/− mice before the re-challenge with oxazolone, suggesting that CD18 was not absolutely required for priming of naïve T cells but was indispensable for T-cell extravasation (
      • Grabbe S.
      • Varga G.
      • Beissert S.
      • et al.
      Beta2 integrins are required for skin homing of primed T cells but not for priming naive T cells.
      ). Hence, 2–16% of CD18 gene expression proved to be sufficient for oxazolone-specific T cells to emigrate from blood vessels during allergic contact dermatitis (
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). Given that in psoriasis vulgaris specific T cells are most likely directed against an, to date, undefined epidermal antigen, our finding that T cells cannot efficiently extravasate into the skin in CD18−/− mutants may explain the absence of a psoriasiform phenotype in these mice. These findings support the conclusion that the pathogenic involvement of CD4+ T cells in the skin disorder of the CD18hypo PL/J mice depends on a gene-dose effect of CD18 expression.
      C57BL/6 or 129Sv mice with the identical CD18hypo mutation leading to the psoriasiform dermatitis in PL/J mice do not develop an inflammatory skin disease (
      • Bullard D.C.
      • Scharffetter-Kochanek K.
      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ;
      • Barlow S.C.
      • Collins R.G.
      • Ball N.J.
      • Weaver C.T.
      • Schoeb T.R.
      • Bullard D.C.
      Psoriasiform dermatitis susceptibility in Itgb2(tm1Bay) PL/J mice requires low-level CD18 expression and at least two additional loci for progression to severe disease.
      ;
      • Kess D.
      • Peters T.
      • Zamek J.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). These data indicate that apart from the CD18hypo mutation, other gene loci are responsible for the development and the onset of this inflammatory skin disease. In order to further identify involved chromosome fragments, backcross analysis between the susceptible PL/J strain and the resistant C57BL/6 strain was performed. This cross resulted in an F1 generation with no obvious phenotype. When crossing back mice of the F1 generation to the susceptible CD18hypo PL/J mice, 30% of the resulting N2 generation revealed a psoriasiform phenotype, suggesting that apart from the CD18hypo mutation other modifier genes were required for the development of this phenotype (
      • Bullard D.C.
      • Scharffetter-Kochanek K.
      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ;
      • Barlow S.C.
      • Collins R.G.
      • Ball N.J.
      • Weaver C.T.
      • Schoeb T.R.
      • Bullard D.C.
      Psoriasiform dermatitis susceptibility in Itgb2(tm1Bay) PL/J mice requires low-level CD18 expression and at least two additional loci for progression to severe disease.
      ). In order to analyze the critical chromosome fragments of the two mouse strains involved in the precipitation of the phenotype, a genome-wide microsatellite analysis was performed from genomic DNA of the N2 backcross generation. Collectively, the assessment of the LOD scores for the linkage of specific chromosome regions with the disease revealed that, in addition to the CD18 mutation, two genes were found to determine the susceptibility and four or more genes determine the severity of the psoriasiform dermatitis in CD18hypo PL/J mice. Most prominent linkage (LOD score >5) was found for gene loci on chromosome 10, revealing a strong linkage for the incidence of the psoriasiform dermatitis, and on chromosome 6 for the time point of onset of the disease. In addition, female mice were observed to be more susceptible for the psoriasiform skin inflammation than male mice (data submitted for publication). Currently, the generation of congenic strains and positional cloning is now underway to finally identify the genes involved in the psoriasiform skin disease and their potential interaction with CD18.
      The herein summarized data on the role of β2 integrins in a murine model of psoriasis in CD18hypo PL/J mice may further support the conception of T cell-dependent skin inflammation and have relevance also for the understanding of polygenic human inflammatory skin diseases.

      Conflict Of Interest

      The authors state no conflict of interest.

      ACKNOWLEDGMENTS

      We are grateful to Dr Arthur Beaudet and his co-workers (Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX) for the support and enthusiasm to use a genetic approach for dissecting complex phenotypes. This work was supported by the individual research grants of the DFG SCHA 411/12-1, the grant SFB 497-C7, and a grant to the Interdisciplinary Center for Clinical Research (IZKF 01 KS9605 TP E8), both at the University of Ulm, and the grant BMBF 01 KS 9502 at the Center for Molecular Medicine (ZMMK-TV60), University of Cologne.

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